Salmonella infections are a major health problem worldwide. Salmonella causes disease by expressing genes located on pathogenicity islands- Salmonella Pathogenicity Island-1 (SPI-1) genes enable Salmonella to adhere and invade epithelial cells, whereas SPI-2 genes are required for systemic infection. Type three secretory systems (TTSS) are encoded on each pathogenicity island and enable Salmonella to secrete effector molecules into the host, promoting pathogenesis. The present proposal focuses on the regulators that control SPI-2 gene expression. The SPI-2-encoded two-component system SsrA/B is essential for activation of genes encoding the TTSS, as well as virulence factors (effectors) secreted through it. Our previous results identified SsrA/B as non-canonical in that SsrA and SsrB were uncoupled from one another. SsrB was expressed constitutively, while the SsrA kinase was acid-induced. Our most recent work identified a role for unphosphorylated SsrB in de-repressing H-NS from the biofilm global regulator csgD, stimulating biofilm formation. Thus, SsrB sits at the top of a decision point in the switch between two competing Salmonella lifestyles. The phosphorylated form, SsrB~P de-represses H-NS AND activates transcription of SPI-2 genes (virulence, intracellular lifestyle). Unphosphorylated SsrB, available when the SsrA kinase is absent, stimulates biofilm formation (carrier state, extracellular lifestyle). Our researh is focused on understanding this switch in molecular terms. The SsrA/B system possesses additional complexity in that two different response regulators control it: ssrA is regulated by OmpR~P, ssrB is regulated by PhoP~P. We recently established that the EnvZ kinase senses the acidic cytoplasm of Salmonella as it resides in the macrophage vacuole; the cytoplasmic domain of EnvZ (EnvZc) was sufficient for sensing. In this proposal, we will test the hypothesis that SsrAc may also respond to the acid cytoplasm to activate SsrB. We will identify whether there are additional genes regulated by SsrB compared to SsrB~P by performing microarrays in the absence and presence of SsrB, ssrB and ssrA null strains and the phosphorylation mutant SsrBD56A. Determining when (and where) SsrA and SsrB are present is the first step towards understanding their regulation. We will examine the appearance, disappearance and localization of SsrA/B by super-resolution imaging and single-particle tracking. We recently showed that the SPI-2 TTSS is present at one/cell and is localized near the cell poles. Super-resolution will reveal whether the SsrA/B system is in close proximity to the SPI-2 secretion system or not. Single particle tracking experiments with SsrB (and SsrB~P) will determine the bound/free ratios and we will quantify the number of SsrA and SsrB molecules present before and during SPI-2 induction by acid. These studies will provide an enhanced understanding of molecular events that activate SsrA/B, a pivotal event required for Salmonella to survive in the host macrophage or drive biofilm formation.